Suspended particle populations found in the water, environmental and medical applications are often composed of many different inorganic, organic and mixed sub-populations. Among these, certain biological species are of particular concern as indicators of pathogen presence or of contamination.
Fluorescent tagging methods are commonly used to identify and count biological species. Fluorescent tags (also called probes) are fluorescent materials, which attach selectively to the entity of interest. Since the amount of the probe which is attached to a single entity, e.g. a bacteria, is small, the fluorescence intensity which can be obtained from one bacteria is also small. In standard methods, the sample is filtered, the filter is placed in a growth medium for the target species, and incubated for many hours allowing the live species to multiply to colonies having a sufficient number of bacteria. The fluorescent probe when applied will attach only to colonies of the target species, which colonies, when present, are observed using fluorescence microscopy, either visually or with a camera, and counted.
Because of the time required for incubation, and because many species can't be cultured, alternative methods attempt to identify and count single target species without the multiplication step.
One method is to apply the fluorescent probe to the sample before or after filtering using a flat filter suitable for microscopic examination. The filter is placed in a fluorescence microscope and illuminated at the fluorescence pumping wavelength. Fluorescent emission of the target species, as identified by an operator either visually or by means of a camera, is used for identification and counting. Provided the microscope is suitably equipped, manual microscopic techniques also allow non-fluorescent images of the same particles to be collected. Unfortunately, these methods are still relatively slow, i.e. taking seconds per particle.
Automatic instruments, which can be used to identify and count sample particles, are of significant practical importance because microscopic analysis requires high skill level and is extremely time consuming, particularly when the concentration of the target species within the overall population is small.
In a conventional automatic instrument, the filter is automatically scanned with an intense optical beam at the probe absorption wavelength. A fluorescence detection system simultaneously examines the point of illumination and detects and counts any fluorescent particles.
U.S. Pat. No. 6,139,800 issued Oct. 31, 2000 to Chandler, and U.S. Pat. No. 6,549,275, issued Apr. 15, 2003 to Cabuz et al disclose another method commonly used to achieve species identification, i.e. flow cytometry, e.g. flow microfluorimetry or flow cytofluorometry. Flow cytometry includes a labeling step, in which target entities within the mixed population are tagged with one or several fluorescent probe compounds that selectively attach only to the target entities. The total particle population is suspended in a transparent liquid carrier. The sampling system is designed so that the particles pass, one at a time, through a small optical excitation zone, which is illuminated with one or more wavelengths. In order to maximize the number of particles analyzed, rapid flow rates of sample liquid are used, e.g. approximately 1 meter per second. Based on measurements of the characteristic scattering and fluorescent light “signature” of each particle, the instrument attempts to identify and resolve the total population into subpopulations. Flow cytometry identifies and classifies particles based on only three parameters, i.e. forward scattering, which represents particle size; side scattering, which represents a combination of surface properties and internal structure; and the presence or absence of a tag attached to the particle. This technique only works well if each of the target particles provides resolvably different signals for one or more of these measurements.
A number of limitations exist with fluorescence tagging methods in the analysis of natural samples, including: selective fluorescent probes are not available or possible for all species; the target entity may not provide a sufficiently distinct optical fluorescence and scattering signature for differentiating from other species; and there may be difficulties in preventing the fluorescent probe compound from attaching to one or more of the wide variety of species, other than the target species, contained within such samples. As a consequence, additional sophisticated techniques, such as immuno-magnetic separation, are required to concentrate the target species before fluorescence analysis is performed.
While flow cytometry does utilize particle morphology information, as derived from scattering signals, the information contained in these signals is limited, especially for particles greater than approximately 5 microns. An alternative existing technology uses only morphological information, derived from image analysis, to differentiate particles. Instruments, which use this method, route samples through a flow cell where a digital camera captures high quality images of each particle on a pixel array. Unfortunately, depth positioning of the particles must be within a few microns, because the allowed depth of focus is very small for high-magnification, high-quality images, typically 3.5 microns for a X10 objective. The system software attempts to classify each particle based on morphological characteristics such as shape, contrast or color. This method can be successful only if the sub-populations of interest are sufficiently different in these characteristics to be resolved by the instrument.
Manual microscopic techniques enable both fluorescent and non-fluorescent images of individual particles to be used in particle identification; however, automatic techniques, which use both types of images, do not exist. Up until now, considerable limitations in the processing rate and system design, which a simultaneous requirement for high quality image formation would normally impose, have prevented such a system from existing. For example: recording and analyzing the signals from each of a large number of detectors, e.g. a minimum of 200 pixels in an array used for high quality imaging, requires much more time than that required to detect and process signals from a small number of individual detectors used for fluorescence and scattering analysis. Furthermore, for accurate image formation, the particle velocity must be sufficiently low so that no significant motion takes place during exposure. Both of these factors limit the rate of particle analysis much below the rate, which can be employed for fluorescence and scattering analysis alone.
Moreover, in order to form a highly magnified image of a particle using standard microscopic techniques, the particle must be located with much higher precision than that required for fluorescence and scattering analysis, because the particle must lie within the depth of focus of the magnification system, e.g. 3.5 microns for a X10 objective. The sample capillary must also provide a clear undistorted optical path with the sample flow placed, with micron accuracy, at the best working distance of the magnification system. These requirements impose significant restrictions on instrument design, which will further reduce performance in the fluorescence and scattering mode relative to an instrument designed solely for this mode.
An object of the present invention is to overcome the shortcomings of the prior art by providing an automatic particle detection system utilizing both fluorescent and non-fluorescent images of individual particles, which are used to measure a larger number of parameters for each particle.